Determination Of Active Ingredients In Lysimachia Clethroides Duby And Study On The Effects On CYP2E1 And CYP3A4 Avtivity

This thesis consists of three chapters. The first chapter summarized the progress of biological activity of flavonoids and the effects on CYP2E1 and CYP3A4, the application of ionic liquids in extraction of flavonoids. The second chapter determinated quercetin, kaempferol, isoquercitrin and astragalin in L. clethroides using ionic liquid-assisted extraction combined with RP-HPLC, and compared the four flavonoids in roots, stems, leaves and flowers of L. clethroides. The third chapter investigated the effects of quercetin, kaempferol, isoquercitrin and astragalin in Lysimachia clethroides Duby on CYP2E1 and CYP3A4, explored the hepatoprotective mechanisms of L. clethroides.Chapter 1 The research progress of flavonoids compoundThe progress of biological activity of flavonoids and the effects on CYP2E1 and CYP3A4, the application of ionic liquids in extraction of flavonoids were summarized.Chapter 2 Analysis of four flavonoids in Lysimachia clethroides Dubyusing ionic liquid-assisted extractionThis chapter established a method for simultaneous determination of isoquercitrin, astragaline,quercetin and kaempferol in L. clethroides using the ionic liquid as the extraction solvent and ultrasound-assisted extraction combined with RP-HPLC. The extraction conditions were optimized by single factor variable method, under the optimized conditions, the mass fraction of isoquercitrin, astragaline,quercetin and kaempferol were 815, 741, 45.3 and 26.5 ?g·g^-1, respectively.The isoquercitrin, astragaline, quercetin and kaempferol in roots, stems, leaves and flowers of L.clethroides were compared. The results showed that the content of the four flavonoids were different, and the contents of isoquercitrin, astragaline in leaves and quercetin, kaempferol in flowers were the highest,respectively. Therefore, the extraction conditions of isoquercitrin, astragaline in leaves and quercetin,kaempferol in flowers of L. clethroides were optimized by single factor variable method. The mass fraction of isoquercitrin, astragaline in leaves and quercetin, kaempferol in flowers were 1.262, 1.219, 0.061 and0.046 mg·g^-1, respectively.The antioxidant activities of methanol and ionic liquid sample solutions of leaves and flowers were evaluated by the DPPH, ABTS, and FRAP assay comprehensively. The results showed that the ionic liquid had no antioxidant activity, and the ionic liquid extract had stronger antioxidant activity than that of the methanol extract.Chapter 3 Effects of flavonoids in Lysimachia clethroides Duby onCYP2E1 and CYP3A4 activities in rat liver microsomesThe incubation systems were established to investigate the effects of quercetin, kaempferol,isoquercitrin and astragalin in L. clethroides on CYP2E1 and CYP3A4 in rat liver microsomes in vitro.Probe drugs of 4-nitrophenol and testosterone as well as the different concentrations of flavonoids were added to incubation systems. After incubation, validated high performance liquid chromatography?HPLC?methods were applied to the separation and determination of the relevant metabolites. The results suggested that kaempferol exhibited weak inhibition against the activity of CYP2E1 with an IC₅₀ of 60.26±2.54 ?M,quercetin and kaempferol had moderate inhibition on CYP3A4 with IC₅₀ values of 18.77±1.69 and32.65±1.32 ?M, but isoquercitrin and astragalin had no effect on the activities of CYP2E1 and CYP3A4.Results indicated that quercetin and kaempferol inhibitory effects were the mechanisms of L. clethroides protecting the liver injure cells. The inhibitory activity of CYP2E1 and CYP3A4 colud inhibit biotransformation of CCl₄ and prevent the production of liver damage material to achieve the role of liver protection.